Methods for introducing mannose 6 phosphate and other oligosaccharides onto glycoproteins and applications thereof

Abstract

Methods to introduce highly phosphorylated mannopyranosyl oligosaccharide derivatives containing mannose-6-phosphate (M6P), or other oligosaccharides bearing other terminal hexoses, to carbonyl groups on oxidized glycans of glycoproteins while retaining their biological activity are described. The methods are useful for modifying glycoproteins, including those produced by recombinant protein expression systems, to increase uptake by cell surface receptor-mediated mechanisms, thus improving their therapeutic efficacy in a variety of applications.

What is claimed is: 
     
       1. A method of coupling a beta-glucocerebrosidase to an oligosaccharide that comprises a phosphorylated mannose, comprising:
 (a) derivatizing an oligosaccharide comprising a phosphorylated mannose with a compound containing a carbonyl-reactive group; 
 (b) oxidizing a beta-glucocerebrosidase having at least one oligosaccharide to generate at least one carbonyl group on the at least one oligosaccharide of the beta-glucocerebrosidase; and 
 (c) reacting the derivatized oligosaccharide with the oxidized beta-glucocerebrosidase, thereby coupling the oligosaccharide to the beta-glucocerebrosidase. 
 
     
     
       2. The method of  claim 1 , wherein the oligosaccharide is a biantennary mannopyranosyl oligosaccharide. 
     
     
       3. The method of  claim 2 , wherein the biantennary mannopyranosyl oligosaccharide comprises bis-mannose 6 phosphate (M6P). 
     
     
       4. The method of  claim 1 , wherein the carbonyl-reactive group is a hydrazine, a hydrazide, an aminooxy, or a semicarbazide. 
     
     
       5. The method of  claim 1 , wherein periodate or galactose oxidase is used to oxidize the beta-glucocerebrosidase. 
     
     
       6. The method of  claim 5 , wherein less than or equal to about 10mM periodate is used to oxidize one or more sialic acid residues on the beta-glucocerebrosidase. 
     
     
       7. A conjugate comprising a beta-glucocerebrosidase coupled to an oligosaccharide comprising a phosphorylated mannose, prepared by the method of  claim 1 . 
     
     
       8. A method of treating Gaucher disease, comprising administering to a subject in need thereof the conjugate of  claim 7 . 
     
     
       9. The method of  claim 8 , wherein the oligosaccharide is a biantennary mannopyranosyl oligosaccharide. 
     
     
       10. The method according to  claim 9 , wherein the biantennary mannopyranosyl oligosaccharide comprises bis-M6P. 
     
     
       11. The method of  claim 8 , wherein the oxidizing is by contact with periodate or galactose oxidase. 
     
     
       12. The method of  claim 8 , wherein the carbonyl-reactive group is a hydrazine, a hydrazide, an aminooxy, or a semicarbazide. 
     
     
       13. A method of coupling a beta-N-acetyl-hexosaminidase to an oligosaccharide that comprises a phosphorylated mannose, comprising:
 (a) derivatizing an oligosaccharide comprising a phosphorylated mannose with a compound containing a carbonyl-reactive group; 
 (b) oxidizing a beta-N-acetyl-hexosaminidase having at least one oligosaccharide to generate at least one carbonyl group on the at least one oligosaccharide of the beta-N-acetyl-hexosaminidase; and 
 (c) reacting the derivatized oligosaccharide with the oxidized beta-N-acetyl-hexosaminidase, 
 thereby coupling the oligosaccharide to the beta-N-acetyl-hexosaminidase. 
 
     
     
       14. The method of  claim 13 , wherein the oligosaccharide is a biantennary mannopyranosyl oligosaccharide. 
     
     
       15. The method of  claim 14 , wherein the biantennary mannopyranosyl oligosaccharide comprises bis-mannose 6 phosphate (M6P). 
     
     
       16. The method of  claim 13 , wherein the carbonyl-reactive group is a hydrazine, a hydrazide, an aminooxy, or a semicarbazide. 
     
     
       17. The method of  claim 13 , wherein periodate or galactose oxidase is used to oxidize the beta-N-acetyl-hexosaminidase. 
     
     
       18. The method of  claim 17 , wherein less than or equal to about 10 mM periodate is used to oxidize one or more sialic acid residues on the beta-N-acetyl-hexosaminidase. 
     
     
       19. A conjugate comprising a beta-N-acetyl-hexosaminidase coupled to an oligosaccharide comprising a phosphorylated mannose, prepared by the method of  claim 13 . 
     
     
       20. A method of treating Tay-Sachs disease, comprising administering to a subject in need thereof the conjugate of  claim 19 . 
     
     
       21. The method of  claim 20 , wherein the oligosaccharide is a biantennary mannopyranosyl oligosaccharide. 
     
     
       22. The method according to  claim 21 , wherein the biantennary mannopyranosyl oligosaccharide comprises bis-M6P. 
     
     
       23. The method of  claim 20 , wherein the oxidizing is by contact with periodate or galactose oxidase. 
     
     
       24. The method of  claim 20 , wherein the carbonyl-reactive group is a hydrazine, a hydrazide, an aminooxy, or a semicarbazide.